Cingulin, Paracingulin, and PLEKHA7: Signaling and Cytoskeletal Adaptors at the Apical Junctional Complex

Ann. N.Y. Acad. Sci. ISSN 0077-8923

ANNALS OF THE NEW YORK ACADEMY OF SCIENCES Issue: Barriers and Channels Formed by Tight Junction Proteins

Cingulin, paracingulin, and PLEKHA7: signaling and cytoskeletal adaptors at the apical junctional complex

Sandra Citi, Pamela Pulimeno, and Serge Paschoud Department of Molecular Biology, University of Geneva, Geneva, Switzerland

Address for correspondence: Sandra Citi, Department of Molecular Biology, 4 Boulevard d’Yvoy, 1211–4 Geneva, Switzerland. [email protected]

Cingulin, paracingulin, and PLEKHA7 are proteins localized in the cytoplasmic region of the apical junctional complex of vertebrate epithelial cells. Cingulin has been detected at tight junctions (TJs), whereas paracingulin has been detected at both TJs and adherens junctions (AJs) and PLEKHA7 has been detected at AJs. One function of cingulin and paracingulin is to regulate the activity of Rho family GTPases at junctions through their direct interaction with guanidine exchange factors of RhoA and Rac1. Cingulin also contributes to the regulation of transcription of several genes in different types of cultured cells, in part through its ability to modulate RhoA activity. PLEKHA7, together with paracingulin, is part of a protein complex that links E-cadherin to the microtubule cytoskeleton at AJs. In this paper, we review the current knowledge about these proteins, including their discovery, the characterization of their expression, localization, structure, molecular interactions, and their roles in different developmental and disease model systems.

Keywords: cingulin; paracingulin; PLEKHA7; ZO-1; p120ctn; junctions

features of TJs, confirming that TJ proteins, together Cingulin and paracingulin with AJ proteins, can be part of atypical junctional Cingulin was discovered as a Mr 140 kDa pro- structures. tein that copurified with myosin II from intesti- The sequence of cingulin, its biochemical behav- nal epithelial cells and was localized in an apical ior, and rotary shadowing electron microscopy show circumferential “belt” in these cells1—hence the that the molecule exists as a parallel homodimer of name cingulin, from the Latin cingere (to form a two subunits, each comprising a globular head do- belt around). Immunoelectron microscopy demon- main, a coiled-coil “rod,” and a small globular tail strated that cingulin is localized at the cytoplasmic (Fig. 1).10,11 Although such domain organization is surface of tight junctions (TJs) in intestinal epithe- similar to that of myosin II, the sequence of cingulin lial cells.1,2 Furthermore, the subcellular localization does not indicate either the presence of an actin- and tissue distribution of cingulin indicate a close activated MgATPase activity or a propensity of the correlation with the continuous TJ belt of differen- coiled-coil rod to form higher order multimolecular tiated epithelia2–4 and endothelia,2,5 as well as the assemblies, such as myosin.11 Cingulin binds to and continuous and discontinuous junctions of strat- bundles actin filaments in vitro and also interacts ified epithelia.2,6,7 Interestingly, expression of cin- in vitro with myosin, suggesting that it could link gulin can be induced in fibroblasts by differentiating TJ proteins to the actin cytoskeleton.10,12 However, agents, resulting in its targeting to spot-like adherens since no major changes in the organization of the junctions (AJs).8 Moreover, cingulin is detectable at actincytoskeletonhavebeendetectedincingulin- sites, such as the kidney glomerular slit diaphragms9 depleted cells, it seems that cingulin does not play and the outer limiting membrane of the retina,2 a significant role in controlling the architecture of which do not have the characteristic morphological actin filaments.

doi: 10.1111/j.1749-6632.2012.06506.x Ann. N.Y. Acad. Sci. 1257 (2012) 125–132 c 2012 New York Academy of Sciences. 125 Cingulin, paracingulin, and PLEKHA7 Citi et al.

Figure 1. A molecular machinery linking TJs to AJs. Simpliﬁed diagram representing the molecular interactions between cingulin (CGN), paracingulin (CGNL1), PLEKHA7, the TJ protein ZO-1, and the AJ protein p120ctn. The TJ and AJ domains of the plasma membrane are represented on the left, with schematics of JAM-A (purple) and claudins/occludin (blue) at TJs and E-cadherin (blue) at AJs. The major structural domains of the proteins are illustrated by colored boxes, whereas the regions involved in mutual interactions are highlighted by red rectangles, linked by lines with arrows. The head and rod domains are shown for CGN and CGNL1, with the ZIM (ZO-1–interaction motif) regions in the N-terminal part of the head,16,30 and the PLEKHA7-interacting region C-terminal to the ZIM in CGNL1.16 Dotted lines above the schemes of CGN and CGNL1 indicate the regions that interact with GEFs in vitro.21,22,27 ZO-1 contains PDZ domains that mediate the interaction with transmembrane proteins, Src homology 3 (SH3), guanylate kinase (GUK), and actin-binding-region (ABR) domains.33 The CGN- and CGNL1-binding region of ZO-1, as identiﬁed by yeast two-hybrid screens, is composed within the region C-terminal to the ABR. The CGNL1- and p120ctn-interacting regions of PLEKHA7 are distinct and located in the central region of the molecule. p120ctn contains a central region with armadillo (ARM) repeats. Afadin is another key component of the apical AJ belt but has been omitted from this scheme for the sake of clarity. [Correction added July 17, 2012 after online publication: Figure has been changed to show the correct dotted lines above the scheme of CGN]

Paracingulin (Mr 150–160 kDa) is likely to be rescence shows junctional and apical localizations, a paralogue of cingulin—e.g., the two proteins whereas immunoelectron microscopy shows an ex- probably arose from a gene duplication event clusive TJ localization. Furthermore, in intestinal and hence the name “para”-cingulin.13,14 Cingulin tissue, paracingulin is associated with nonjunctional and paracingulin show a good degree of sequence actin filaments in the basal region of the cells.15 homology, especially in the coiled-coil rod do- A nonjunctional localization of paracingulin along main (39% identity) and in the N-terminal ZO-1– actin stress fibers has also been observed in fibrob- interacting motif (ZIM; 73% identity; Fig. 1).13,15,16 lasts expressing exogenous paracingulin.15,16 There- Thus, we speculate that paracingulin also exists as fore, although the actin- and myosin-binding prop- a parallel dimer. The head region of paracingulin erties of paracingulin are not known, paracingulin is larger than the head domain of cingulin (ap- may have a more direct association with the actin proximately 600 versus approximately 350 residues, cytoskeleton than cingulin. depending on species), and the coiled-coil rod is Unlike cingulin, paracingulin is structurally smaller (approximately 660 residues versus approx- linked to the microtubule cytoskeleton. Perturb- imately 800), accounting for the difference in their ing the organization of microtubules results in apparent molecular size. Paracingulin was inde- loss of junctional paracingulin, whereas cingulin pendently characterized as a junction-associated is unaffected.14 This is probably due to the inter- coiled-coil protein (JACOP).15 Immunoelectron action of paracingulin with PLEKHA7,16 which is microscopy analysis showed that unlike cingulin, indirectly bound to microtubules17 (see later). In the localization of paracingulin is more promiscu- contrast, both cingulin and paracingulin localiza- ous. In kidney tissue, where paracingulin mRNA tions are dramatically affected by actin filament- is detected at high levels, paracingulin is localized disrupting drugs,14 confirming the association of both at TJs and AJs.15 In liver tissue, immunofluo- both proteins with the actin cytoskeleton. Indeed,

126 Ann. N.Y. Acad. Sci. 1257 (2012) 125–132 c 2012 New York Academy of Sciences. Citi et al. Cingulin, paracingulin, and PLEKHA7 the dynamics of junction exchange of cingulin and of cingulin does not have any detectable effect on paracingulin are similar to actin and more rapid junction assembly and development of the TJ bar- than those of ZO-1.14 Cingulin and paracingulin rier in kidney cells, and the junctional targeting and can be detected in a complex together, and with activity of p114RhoGEF in these cells depends on otherTJproteins.14,16 However, since the junctional the FERM domain–containing protein Lulu2, rather recruitment and dynamics of cingulin are indepen- than cingulin.24 dent of paracingulin and vice-versa, the two pro- Significantly, the increase in cell proliferation, teins likely do not function as a unit, but rather cell density at confluence, and claudin-2 expres- as independent proteins, with partially redundant sion in cingulin-depleted kidney epithelial cells are functions.14 rescued by the inhibition of RhoA, indicating that In summary, cingulin and paracingulin are struc- RhoA mediates, at least in part, the effects of cin- turally homologous proteins, with an asymmetric gulin on transcriptional and cell cycle regulation.22 shape, similar dynamics, partially overlapping sub- It is important to note that RhoA is modulated by cellular localizations, and distinct interactions with several other junctional molecules,25 and that tran- the actin and microtubule cytoskeletons. scription factors such as ZONAB and YAP are putative downstream effectors of RhoA.26 Therefore, Cingulin and paracingulin as adaptors for the activity of cingulin as a RhoA regulator must guanidine exchange factors be viewed in the context of a wider signaling net- The first direct approach to study the function of work that is likely to exist in different cell-specific cingulin was to generate embryonic stem (ES) cells configurations. lacking cingulin through homologous recombina- Paracingulin also interacts with the RhoA tion. The results showed that embryoid bodies (EB) activator GEF-H1; consequently, depletion of obtained by differentiation of cingulin knockout paracingulin in confluent cells results in increased (KO) ES cells had apparently normal TJs, based RhoA activity, an effect that phenocopies cingulin on immunofluorescence, electron microscopy, and depletion. However, unlike cingulin, paracingulin permeability assays, but had a remarkably altered also interacts with the Rac1 activator Tiam1. This pattern of gene expression: over 800 genes showed results in a delay in the establishment of the greater than twofold change in expression.18,19 Since TJ barrier, reduced peak of transepithelial resis- these genes include several transcription factors, tance, and decreased Rac1 activation during the downstream target genes, and proteins such as process of junction assembly/barrier formation in claudin-2—whose expression is correlated to cell MDCK cells.27 Indeed,Tiam1recruitmentatjunc- differentiation—their alteration indicates that cin- tions of MDCK cells is impaired in paracingulin- gulin is a regulator of signaling pathways that depleted cells, indicating an important role of ultimately affects transcription and differentia- paracingulin in Rac1 activation via the junctional tion. Interestingly, the cingulin promoter has been recruitment of Tiam1.27 Tiam1 has been shown identified as a target of HNF-␣, a transcription to play a key role in Rac1 regulation in differ- factor regarded as a key regulator of intestinal ent cell model systems, to interact with the Par differentiation.20 polarity complex, and to regulate Ras-induced , To address the molecular mechanisms through oncogenesis.28 29 which cingulin regulates gene expression and cell In summary, although it is not clear to what ex- differentiation, we isolated cingulin-depleted kid- tent paracingulin is implicated in the regulation ney epithelial cell clones and discovered that an- of gene expression and cell proliferation, through other effect of cingulin depletion is an increase in RhoA or other mechanisms, the available data in- RhoA activation in confluent monolayers, due to dicate that cingulin and paracingulin contribute to the junctional sequestration of the RhoA activa- fine-tuning the activity of Rho family GTPases both tor GEF-H1.21,22 Cingulin binds directly to GEF- during junction assembly (paracingulin) and at con- H1 in vitro19,22 and has also been implicated in fluence (cingulin and paracingulin). Furthermore, the junctional recruitment of a second Rho GEF, modulation of RhoA activity by cingulin is involved p114RhoGEF, which is important for junction as- in its ability to control cell proliferation and gene sembly in corneal cells.23 However, depletion or KO expression.

Ann. N.Y. Acad. Sci. 1257 (2012) 125–132 c 2012 New York Academy of Sciences. 127 Cingulin, paracingulin, and PLEKHA7 Citi et al.

Identification of PLEKHA7 as a creased expression of paracingulin, indicating that paracingulin-interacting protein PLEKHA7 plays a major role in the recruitment and stabilization of paracingulin to AJ. Indeed, the as- Cingulin and paracingulin are both localized at TJs, sociation of paracingulin with PLEKHA7 at AJs can but paracingulin is also detected at AJs and in associ- explain why ZO-1 depletion only delays, but does ation with extra-junctional actin filaments. What is not significantly decrease, paracingulin junctional the molecular basis for these different localizations? recruitment.16 Recent studies have provided some answers to these The mechanisms controlling subcellular localiza- questions. ZO-1 contributes to TJ recruitment of tion of paracingulin should be investigated in ad- both cingulin and paracingulin, as indicated in ex- ditional cell types, considering the heterogeneous periments showing that in cells depleted of ZO-1, localization of paracingulin in tissues. For exam- but not ZO-2, the junctional recruitment of cin- ple, it is not clear which molecular interactions tar- gulin is decreased and the junctional recruitment 16 get paracingulin to actin stress fibers in epithelial of paracingulin is delayed. Both proteins bind , and nonepithelial cells.15 16 This targeting occurs to ZO-1 in vitro through their conserved ZIM in independently of the interaction with ZO-1– and the N-terminal region of the globular head domain PLEKHA7, since it is observed in Rat-1 fibrob- (Fig. 1), and deletion of the same region abolishes , lasts using exogenous mutant constructs lacking the cingulin junctional targeting in transfected cells.16 30 ZO-1– and PLEKHA7-interacting regions.16 The Thus, it is reasonable to conclude that cingulin and functional role of paracingulin at stress fibers re- paracingulin directly bind to ZO-1 at TJs. However, mains unknown, but considering its ability to inter- despite the observation that in an epithelial breast act with GEFs that regulate Rho family GTPases, a cancer line ZO-1 KO completely abolishes cingulin putative role in regulating RhoA and Rac1 activities junctional localization,31 a normal junctional lo- at this site could be hypothesized. calization of cingulin was observed in tissues from 32 ZO-1 KO mouse embryos. Therefore, the mecha- PLEKHA7: a new component of the apical nisms of cingulin junctional recruitment appear to AJ belt be cell-context dependent, and redundant interactions can target cingulin, and possibly also paracin- PLEKHA7 was discovered independently by Take- gulin, to TJs. Indeed, in vitro cingulin binds not ichi’s and our laboratory as a novel protein , , only to ZO-1 and ZO-2 but also to ZO-3 and other component of epithelial AJs.16 17 34 The distinguish- TJ proteins.10 Interestingly, the region of ZO-1 suffi- ing feature of PLEKHA7, with respect to most cient for interaction with either cingulin or paracin- other AJ proteins, except afadin, is that it is con- gulin (by the yeast two-hybrid technique) is the fined to a continuous apical belt and is absent C-terminal region adjacent to the actin-binding re- from the lateral membrane.34 In the pancreas, gion (Fig. 1). Thus, the C-terminal region of ZO-1 for example, p120ctn and PLEKHA7 labeling are appears to be a structural module dedicated to inter- only partially overlapping, with PLEKHA7 strongly action with actin, actin-binding proteins, and other labeling ducts, whereas p120ctn is localized along cytoskeletal proteins.33 both junctional and lateral surfaces of exocrine aci- A yeast two-hybrid screen revealed PLEKHA7 as nar cells (Fig. 2). The localization of PLEKHA7 in a very high-confidence interactor of paracingulin; several tissues is highly reminiscent of the localiza- and we showed that the AJ targeting of paracingulin tion of TJ proteins and similar to that of afadin,34 in MDCK cells is mediated by its interaction with another AJ protein that has a belt-like distribution PLEKHA7.16 PLEKHA7 is an AJ protein34 compris- and has alternatively been described as a TJ pro- , ing WW, proline-rich, PH, and coiled-coil domains tein.35 36 Double labeling of PLEKHA7 with ZO-1 (Fig. 1). An N-terminal region of the head domain shows a partial co-localization, which may depend of paracingulin, distinct from the ZO-1–interacting on tissue type and section orientation (Fig. 2). How- region, interacts with a region in the C-terminal ever, the overall localization and tissue distribution domain of PLEKHA7, the first coiled-coil domain16 of ZO-1 and PLEKHA7 are not identical, and im- (Fig. 1). Depletion of PLEKHA7 in kidney epithelial munoelectron microscopy of intestinal cells shows cells results in a loss of junctional staining and de- a concentration of PLEKHA7 labeling at AJs.34

128 Ann. N.Y. Acad. Sci. 1257 (2012) 125–132 c 2012 New York Academy of Sciences. Citi et al. Cingulin, paracingulin, and PLEKHA7

membrane domain, results in the redistribution of both p120ctn and PLEKHA7, indicating that PLEKHA7 may also be associated with N-cadherin based junctions, through p120ctn.17 However, the observation that PLEKHA7 is only associated with p120ctn along the AJ belt, but not along the lateral surfaces of polarized epithelial cells (Fig. 2),34 suggests that additional interactions are essential to target PLEKHA7 to the junctional belt. Cingulin and PLEKHA7 in development and disease Insights about the functions of junctional proteins can be obtained from studies about their roles in development and disease. Cingulin is maternally expressed and cortically localized both in mouse and in Xenopus laevis embryos.37–40 In the mouse early embryo, cytocortical maternal cingulin is degraded by endocytic turnover, and zygotically expressed cingulin accumulates at TJs after the 16-cell stage, just after ZO-1.37,38 Moreover, cingulin is upregulated in the mouse blastocyst trophectoderm, and its stabil- ity requires cell–cell contact.38 In the frog embryo, maternal cingulin is recruited from the apical cor- Figure 2. The localization of PLEKHA7 at the apical AJ belt. tex into the newly forming TJs starting from the first Double immunofluorescent localization of PLEKHA7 (red) and cleavage, and defines the border between apical and p120ctn (green) in frozen sections of pancreas (top), and of ZO- 39,40 1 (red) and PLEKHA7 (green) in frozen sections of duodenum. lateral membranes. Depletion or KO of cingulin Samples were obtained and processed as described.34 The boxed from either Xenopus embryos or mice, using either areas in the low magnification images are shown enlarged below. morpholino oligos or gene targeting techniques, re- Arrowheads indicate lack of co-localization, and arrows indicate spectively, does not impair embryo viability (our co-localization. Nuclei are labeled in blue by DAPI in merged unpublished results). However, it was recently re- images. Bar = 2 µm. Note that PLEKHA7 is not co-localized with p120ctn along the lateral surface of acinar epithelial cells ported that morpholino-mediated depletion of cin- (see also Ref. 34) but co-localizes with p120ctn along intralob- gulin from the developing neural crest of the chick ular and interlobular ducts. ZO-1 is not highly expressed in the midbrain expands the size of the migratory neural duodenum, but in some sections of cells expressing sufficient crest cell domain.41 Moreover, in the chicken em- amounts of ZO-1, there is a partial co-localization of ZO-1 with bryo, overexpression of cingulin is correlated with PLEKHA7, indicating that the apical, PLEKHA7-containing AJ belt is immediately adjacent to the ZO-1–containing TJ belt. changes in RhoA protein distribution in ventrolat- eral neuroepithelial cells, although it is not clear whether RhoA activity is altered in this model.41 In- Besides recruiting paracingulin to kidney epithe- terestingly, overexpression of either full-length cin- lial AJs, PLEKHA7 is part of a molecular com- gulin or its domains does not result in changes to plex linking E-cadherin to microtubules, through RhoA activity in cultured cells.42 Nevertheless, the p120ctn and nezha, and stabilizing AJs.17 PLEKHA7 observations on chick embryos41 suggest a possible was indeed identified as a protein binding to the role of cingulin in regulating neuroepithelial differ- N-terminal domain of p120ctn, and which is re- entiation and neural crest development. quired to maintain the junctional localization of Little is known about the role of cingulin in hu- E-cadherin, nezha, and KIFC3, a minus-ended ki- man disease. The expression of cingulin in human nesin motor that binds to nezha.17 In cadherin- cancers indicates that its expression is maintained deficient neuroblastoma cells, the expression of a and sometimes increased in benign and malignant mutant form of N-cadherin, lacking the juxta- adenocarcinomas, but is typically lost in squamous

Ann. N.Y. Acad. Sci. 1257 (2012) 125–132 c 2012 New York Academy of Sciences. 129 Cingulin, paracingulin, and PLEKHA7 Citi et al. carcinomas, whereas it is undetectable in nonep- ized epithelial cells, by orchestrating the formation ithelial tumors.43,44 In undifferentiated adenocar- of a continuous circumferential belt at the apical cinomas, the expression of cingulin and other TJ junctional complex. proteins can be detected in areas bordering the It is noteworthy that homologues of cingulin, stroma, in the absence of a lumen.43,45 Therefore, paracingulin, and PLEKHA7 have not been de- cingulin, together with other TJ proteins, can be scribed in invertebrate organisms. On the one hand, used as an additional marker to identify epithe- this limits the availability of genetic experimental lial tumors, and distinguish adenocarcinomas from tools, but on the other hand, this underlines the squamous carcinomas.44 potential importance of these proteins for the evo- PLEKHA7, on the other hand, has been impli- lution of highly complex signaling networks, typical cated in heart development and hypertension. Sin- of vertebrates. gle nucleotide polymorphisms of the PLEKHA7 locus are associated with diastolic high blood pres- Acknowledgments sure in genome-wide studies on caucasian and asian We thank Rocio Tapia and Domenica Spadaro for ethnic groups.46–48 However, the cellular mecha- comments on the manuscript, and the Swiss Na- nisms whereby mutations in the PLEKHA7 locus tional Foundation, the State of Geneva, and the Sec- lead to increased blood pressure are unknown. tion of Biology of the Faculty of Sciences of the Knockdown studies show that the zebrafish ho- University for financial support. molog of PLEKHA7, Hadp1, is required for cardiac contractility and morphogenesis, through a Conflicts of interest mechanism involving the regulation of intracellular The authors declare no conflicts of interest. Ca2+ handling, and possibly phosphatidylinositols, in the absence of morphological defects.49 However, References the subcellular localization of PLEKHA7/Hadp1 in 1. Citi, S., H. Sabanay, R. Jakes, B. Geiger & J. Kendrick-Jones. 34,49 heart tissue has not been clarified. Therefore, 1988. Cingulin, a new peripheral component of tight junc- PLEKHA7 functions as a cytoskeletal adaptor in ep- tions. Nature 333: 272–276. ithelial cells, and controls cardiac function through 2. Citi, S., H. Sabanay, J. Kendrick-Jones & B. Geiger. 1989. mechanisms that remain to be investigated. Cingulin: characterization and localization. J. Cell Sci. 93: 107–122. Conclusion 3. Halbleib, J.M., A.M. Saaf, P.O. Brown & W.J. Nelson. 2007. Transcriptional modulation of genes encoding structural Cingulin and paracingulin are essential components characteristics of differentiating enterocytes during devel- of a regulatory network controlling the activity of opment of a polarized epithelium in vitro. Mol. Biol. Cell. 18: 4261–4278. Rho family GTPases at the apical junctional com- 4. Erickson, D.R., S.R. Schwarze, J.K. Dixon, C.J. Clark & M.A. plex, and possibly at other cellular sites. This activity Hersh. 2008. Differentiation associated changes in gene ex- may be important to control gene expression, cell pression profiles of interstitial cystitis and control urothelial proliferation, and hence differentiation and mor- cells. J. Urol. 180: 2681–2687. phogenesis, although additional work is required 5. Corada, M. et al. 1999. Vascular endothelial-cadherin is an important determinant of microvascular integrity in vivo. to confirm these functions at the whole organism Proc. Natl. Acad. Sci. USA 96: 9815–9820. level. Furthermore, both proteins are part of mul- 6. Schluter, H., I. Moll, H. Wolburg & W.W. Franke. 2007. timolecular complexes linking cell–cell junctions to The different structures containing tight junction proteins the actin and microtubule cytoskeletons. PLEKHA7, in epidermal and other stratified epithelial cells, including a recently characterized protein, may have multiple squamous cell metaplasia. Eur. J. Cell Biol. 86: 645–655. 7. Langbein, L. et al. 2002. Tight junctions and composition- roles in epithelial and cardiac cells, both as a cy- ally related junctional structures in mammalian stratified 2+ toskeletal linker and a regulator of intracellular Ca epithelia and cell cultures derived therefrom. Eur. J. Cell signaling. Biol. 81: 419–435. We propose that cingulin, ZO-1, paracingulin, 8. Bordin, M., F. D’ Atri, L. Guillemot & S. Citi. 2004. Histone and PLEKHA7, together with afadin and other reg- deacetylase inhibitors up-regulate the expression of tight 50–52 junction proteins. Mol. Cancer Res. 2: 692–701. ulatory and cytoskeletal proteins, are part of a 9. Fukasawa, H., S. Bornheimer, K. Kudlicka & M.G. Farquhar. molecular framework that links TJ to AJ (Fig. 1), 2009. Slit diaphragms contain tight junction proteins. JAm and thus the apical to the lateral domain of polar- Soc Nephrol. 20: 1491–1503.

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